Molecular basis for the temperature-dependent insolubility of cryoglobulins. X. The amino acid sequence of the heavy chain variable region of McE.
The amino acid sequence of the VH region of McE, a monoclonal IgM cryoimmunoglobulin, has been determined employing automated sequencing methodology. Digestion of the intact Fab mu component, derived by trypsin cleavage of the parent protein at elevated temperature with CNBr, followed by complete reduction and alkylation, yielded the intact light chain as well as the 2 CNBr fragments that constituted the VH. N-terminal sequencing of the larger unblocked CNBr fragment, along with a component fragment derived by cleavage by BNPS-Skatole, established the structure of the VH from position 88 through the V leads to C switch region. Citraconylation of the smaller, blocked fragment effected sufficient solubilization for enzymatic deblocking and direct sequencing of the N-terminal 20 residues of the VH. Complete trypsin digestion of the N-terminal CNBr fragment yielded 9 peptides that could be isolated by preparative cation exchange chromatography and gel filtration. The complete sequence of these peptides along with 4 chymotryptic peptides completed the primary structure of the VH region. The primary structure of McE appears to resemble that of He, previously identified as belonging to the VH II subgroup. The presence of characteristic CDR and FR regions as well as the identification of a probable site of glycosylation suggest that the cryoimmunoglobulin closely resembles noncryoglobulins in terms of overall structural composition. The cryoglobulin property may arise through alterations in individual residues or unfavorable arrangements of CDR and FR segments.
D000595:Amino Acid Sequence; D001666:Binding Sites, Antibody; D055598:Chemical Phenomena; D002621:Chemistry; D003450:Cryoglobulins; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007075:Immunoglobulin M; D007135:Immunoglobulin Variable Region; D007148:Immunoglobulin mu-Chains; D012995:Solubility; D013696:Temperature; D014357:Trypsin / Q000494:pharmacology
D003450:Cryoglobulins; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007075:Immunoglobulin M; D007135:Immunoglobulin Variable Region; D007148:Immunoglobulin mu-Chains; D014357:Trypsin